Challenges and Prospects for Designer T and NK Cells in Glioblastoma Immunotherapy

Abstract

Glioblastoma (GBM) is the most prevalent, aggressive primary brain tumour with a dismal prognosis. Treatment at diagnosis has limited efficacy and there is no standardised treatment at recurrence. New, personalised treatment options are under investigation, although challenges persist for heterogenous tumours such as GBM. Gene editing technologies are a game changer, enabling design of novel molecular-immunological treatments to be used in combination with chemoradiation, to achieve long lasting survival benefits for patients. Here, we review the literature on how cutting-edge molecular gene editing technologies can be applied to known and emerging tumour-associated antigens to enhance chimeric antigen receptor T and NK cell therapies for GBM. A tight balance of limiting neurotoxicity, avoiding tumour antigen loss and therapy resistance, while simultaneously promoting long-term persistence of the adoptively transferred cells must be maintained to significantly improve patient survival. We discuss the opportunities and challenges posed by the brain contexture to the administration of the treatments and achieving sustained clinical responses.

Keywords: CRISPR/Cas9; T cells; chimeric antigen receptor; genomic heterogeneity; glioblastoma; immunotherapy; natural killer cells.

Figure 1

Schematic representation of the glioblastoma (GBM) tumour microenvironment. The evolving tumour microenvironment consists of diffusely infiltrative tumour cells with a leaky neovasculature, anti-inflammatory stromal cells and high concentrations of immunosuppressive cytokines and signalling molecules. Together, these factors generate a hostile environment where proliferation and persistence of cytotoxic immune cells are significantly hampered. TAM, tumour-associated macrophage; MDSC, myeloid derived suppressor cell; T_reg, regulatory T cell; NK cell, natural killer cell; TGFβ, transforming growth factor β; PD-1, programmed cell death protein 1; PD-L1, programmed death-ligand 1; CTLA-4, cytotoxic T lymphocyte-associated protein-4; IL-13, interleukin-13; IL-10, interleukin-10; VEGF, vascular endothelial growth factor; MHC I, major histocompatibility complex class I; JAK/STAT, Janus kinase/signal transducers and activators of transcription; RTK, receptor tyrosine kinase; mTOR, mammalian target of rapamycin.

Figure 2

Schematic representation of chimeric antigen receptor (CAR) constructs. The ectodomain of a CAR contains a single-chain variable fragment (ScFv) region, which is derived from the antigen-binding fragment (Fab) of a monoclonal antibody containing a heavy-chain (V_H) linked to a light-chain (V_L), all linked to the transmembrane domain by a hinge region. The ScFv region contains an antigen recognition domain, which is specific for the desired tumour antigen to be targeted. (A) The first three generations of CAR constructs made alterations to the endodomain, which contains the intracellular signalling domain of the zeta (ζ) chain of the T cell receptor (TCR)/CD3 complex, and co-stimulatory signalling domains (CD27, CD28, 4-1BB or OX-40). (B) Later iterations of CARs based on the 2^nd generation CAR endodomain included induction of activating cytokines (4^th generation TRUCK), antigen recognition domains for two antigens (TanCAR) or required the addition of a small molecule, or Lenalidomide, for the dimerization (through zinc finger domains CRBN and IKZF3) and activation of the CAR (ON-switch CAR).

Figure 3

Schematic representation of CRISPR/Cas9 gene editing. The DNA endonuclease Cas9 is introduced to target cells via transduction, transfection or addition of pure Cas9 protein, and complexes with the guide RNA (gRNA). This complex between Cas9 and gRNA searches for a protospacer adjacent motif (PAM) sequence in proximity to the gRNA 20-base sequence. Once bound, Cas9 causes a double-stranded DNA break, and the cell must use either non-homologous end joining or homology-directed repair to fix the break. This results in either wild type DNA, edited DNA, or insertions and deletions (indels) that result in gene knockout.

Figure 4

Combination CAR T cell therapy with gene editing to treat GBM. T cells are isolated from the blood of a cancer patient. CRISPR/Cas9 gene editing technology can then be used to knock out inhibitory receptor PD-1, and viral transduction induces expression of chimeric antigen receptors (CARs) targeting tumour-associated antigens CD73 and CD39. To avoid serious adverse events, inducible Caspase 9 (iCaspase9) can be activated to swiftly eliminate the adoptively transferred cells. The altered T cells can then be expanded and re-introduced to the cancer patient. Image adapted with permission from [111] from AAAS. gRNA, guide RNA; PD-1, programmed cell death protein 1; CD39, cluster of differentiation (CD) 39 (Ectonucleoside triphosphate diphosphohydrolase-1); CD73, (5′-nucleotidase); CD47, (integrin-associated protein).